Summary A role for reactive nitrogen species in aging as well as in over eighty human diseases including atherosclerosis, cancer, chronic pain, infection, neurodegeneration, and stroke has been demonstrated by using 3-nitrotyrosine (nitroTyr) as a biomarker. In these conditions, tyrosine nitration is not randomly distributed, but specific tyrosines on certain proteins are more readily modified. The central hypothesis of this proposal is that nitroTyr-modified proteins are key players in human disease and that understanding their mechanistic role in pathology will lead to new opportunities for therapeutic intervention. The challenge using conventional biochemical and cell- based approaches has been how to determine which nitroTyr modifications are functionally significant and which are inconsequential. The PI has shown that this hurdle can be overcome by using genetic code expansion technology to quantitatively and site-specifically incorporate nitroTyr in a targeted manner into recombinant proteins produced in bacteria. This approach has now been used to provide the first two demonstrations that specific nitroTyr-proteins in a given disease have altered properties that implicate them as key players in the development of pathology. In one case, the nitration of either of two specific tyrosines in heat shock protein 90 (Hsp90) can cause motor neuron death in amyotrophic lateral sclerosis, and in the other case that the nitration of a single Tyr in the protein Apolipoprotein A1 leads to its selective incorporation into atherosclerotic plaques. The next step in facilitating determination of the mechanisms of pathology for nitroTyr-proteins is to be able to encode them in mammalian cells so that one can directly determine in vivo how nitroTyr modifications alter protein function, interactions, and regulation. The focus of this proposal is to pursue two aims that encompass (1) developing the needed tools for mammalian expression of nitroTyr-proteins, and (2) applying the tools to carry out both in vitro and in vivo studies to elucidate the mechanisms by which tyrosine nitration alters protein interactions in a biologically relevant model system of known physiological importance. The selected model system centers on key tyrosines of calmodulin (CaM) and Hsp90, and how their nitration alters calcium regulation of nitric oxide and superoxide production from a common client protein, the endothelial nitric oxide synthase. The tools created will overcome a major roadblock in the field by providing an approach to assess in mammalian cells the functional impacts of specific nitroTyr residues in any given protein. The work will also provide initial insights into the open questions of how nitration at specific tyrosines impacts select functions of CaM and Hsp90, and the interplay between tyrosine nitration and phosphorylation. This work will have a sustained impact by providing a fundamentally new approach that can be used to understand how tyrosine nitration affects disease progression in the many human diseases in which it occurs. And for every case in which it is discovered that nitroTyr formation does contribute to pathology development, the mapping of that process will open up a new avenue for therapeutic intervention.